Thus, it was determined that piR-823 might show different expressions in different type of cancers.PIWI interacting RNAs (piRNAs), a member of non-coding RNA, originate from intergenic repetitive regions of the genome. piRNA expressions increase in various cancers and it is thought that this increase could be caused by hormones. We aimed to determine the effects of hormones on piRNA expression in breast and prostate cancer. High viability and a decrease in adhesion were observed at the concentrations of the highest proliferation. Furthermore, an increase in adhesion was also observed in MDA-MB-231 cells. After hormone treatment, while  Gender dependent steroid hormones play an important role in the development and mechanism of cancer of the reproductive system, particularly in prostate cancer in males and uterus and breast cancer in females ,2. AndroDNMT3, which is a DNA methyltransferase [The importance of non-coding RNAs in the development and prognosis of most diseases, particularly in cancer, has been increasing more and more, and the studies which have been carried out mark their importance as epigenetic regulators ,9. piRNAnsferase ,12–14. Insferase –17. piRNnsferase ,18,19. Gnsferase –22. Thounsferase ,23–25, snsferase .piRNA-651 (piR-651) and piRNA-823 (piR-823), which are among the piRNAs thought to be effective in gender-dependent cancers.Our study investigates the piRNAs in hormone-dependent and hormone-independent cancer cells, and its aim is to determine whether these piRNAs play effective roles only in hormone-dependent cancers or the same is true for hormone-independent cancers as well. For this purpose, we treated androgen-dependent and androgen-independent prostate cancer cells (LNCaP and PC-3) with an androgen hormone, and estrogen-dependent and estrogen-independent breast cancer cells (MCF-7 and MDA-MB-231) with an estrogen hormone to investigate cell proliferation, viability, adhesion and hormone expression. Moreover, the present study identifies the expression of 2 in Dulbecco’s Modified Eagle’s Medium  containing 1% penicillin/streptomycin  and 10% foetal bovine serum (FBS) . Before hormone treatment, the breast and prostate cancer cells were incubated in 6-well plates  to make sure the cells grew to 80% confluence. Estrogen (E2257) and androgen (A0887) were obtained from Sigma . To observe the appropriate concentration, hormones with certain concentration ranges were used to treat cells according to the instructions of the manufacturer. Using the XTT method , the most proliferative concentration and hours were determined to be the 24th, 48th and 72nd hours. By deciding on the optimal concentration of hormones and optimal time, viability, proliferation, adhesion and piRNA expression, assays were applied to these specified conditions.Prostate cancer cell lines (LNCaP and PC-3) and breast cancer cell lines (MCF-7 and MDA-MB-231) were purchased from the American Type Culture Collection , and the cells were incubated at 37°C with 5% COThe cell viability rate was determined using the trypan blue method. Hormone treated cells were exposed to trypan blue  after being trypsinized from a flask. The live and dead cells were counted using the Thoma Slide .2 for 2 hours. The microplate spectrophotometer ) was used to determine the absorbance at 450nm.The XTT Kit  was used to determine the impacts of hormones on the proliferation of breast and prostate cancer cell lines. In brief, the cells were plated into 96-well plates  and, after optimal hormone treatment, XTT was pipetted to all wells and cultured at 37°C with 5% CO2 for 2 hours, and the microplate spectrophotometer ) was used to determine the absorbance at 450nm.The XTT Kit  was again used, this time to determine the impacts of hormones on the adhesion of breast and prostate cancer cell lines, and, as before, the cells were plated into 96-well plates . After optimal hormone treatment, the wells were flushed three times with phosphate-buffered saline . XTT was then pipetted to all wells and cultured at 37°C with 5% COpiR–651, piR–823 and Glyseraldehide-3-phosphate dehydrogenase (GAPDH) were designed and supplied by Alpha DNA, Montreal, Quebec. RT-PCR was carried out inside a Strategene MxPro3000 . GAPDH  was used as an internal control, and the expression of piR–651 and piR–823 was normalized in line with the expression of GAPDH. Gene expression changes were quantified using the delta-delta CT method. The forward and reverse primer sequences used in RT-PCR are shown in Total RNA was isolated from cells using the Paris Total RNA Isolation Kit  in accordance with the instructions of the manufacturer. The RNAs isolated from the hormone treated cells were converted to cDNA through a reverse transcription . Primer sets for the amplification of A normal distribution of the continuous variables was enabled using the Kolmogorov-Smirnov suitability test. Comparisons between groups of normally distributed variables were evaluated using One-Way variance analysis (ANOVA). The Tukey HSD test was used for multiple comparisons. Comparisons between groups of variables which were not normally distributed were evaluated using the Kruskal-Wallis test. Multiple comparisons of these groups were evaluated using the Dunn test, while normally distributed piRNA values were compared using the Student t test. All analyses were carried out using the IBM SPSS Statistics 21.0 software package  Files. TLNCaP androgen-dependent prostate cancer cells were treated with androgen at concentrations of 200nM, 100nM, 50nM, 25nM, 10nM, 1nM, and 0.1nM. Ethanol was used to dissolve the androgen and estrogen hormones in the medium, an additional ethanol group being formed for this reason. In the measurement carried out 24 hours after treatment with the hormone, statistically significant increases were observed in the 10nM androgen group (847229±19949)  and the 1nM androgen group (915657±25069)  when compared with the control group (156729±22278). The PC-3 cells were treated with 10nM, 1nM and 0.1nM androgen, and, when compared with the control group (41961±907), there was a statistically significant decrease in the 1nM androgen group (25990±5334) . The MCF–7 cells were treated with 10nM, 1nM and 0.1nM estrogen, but no statistically significant difference was found between the groups . The MDA-MB–231 cells were treated with 10nM, 1nM and 0.1nM estrogen, but no statistically significant difference was found between the groups .When compared with the control group (156728.57±58943.07), a significant increase in the number of cells in the 1nM androgen group (915657.14±66326.66) was detected in the LNCaP cells (P < 0.001). While cells were detected in the control group (41961.43±2398.41) and in the 10nM androgen group (40561.43±5547.29) of PC-3 cells, but no statistically significant difference was found between the groups (P >0.05). In the MCF-7 cells, an increase was detected in the proliferation of the 10nM estrogen group (97704.71±7623.37) when compared with the ethanol group (82252.29±14261.99) (P < 0.05). In the MDA-MB-231 cells, white cells were detected in the control group (20816.71±7496.41) and 1nM estrogen group (32489.29±10916.76)   of the 1nM androgen group when compared with the control (80.88±9.92) and ethanol groups (68±9.21) (P < 0.001). In the PC-3 cells, while the viable cell count percentage was detected to have increased in the 10nM androgen group (78.75±20.83) when compared to the ethanol group (73.88±19.48), no statistically significant difference was found between the groups (P >0.05). It was observed that the treatment of the MCF-7 cells with estrogen increased the viable cell count percentage of the 10nM estrogen group (86.75±9.54) when compared with the control (82.13±12.41) and ethanol (71.88±8.27) groups (P < 0.05). In the MDA-MB-231 cells, while the viable cell count % was detected to have increased in the 1nM estrogen group (86.13±15.41) when compared with the control (72.88±29.53) and ethanol (77±21.58) groups, no statistically significant difference was found between the groups   when compared with the control group (0.93±0.09) (P < 0.05), while in the PC-3 cells, a statistically significant increase was detected in the adhesion in ethanol (0.86±0.06) and the 10nM androgen group (0.84±0.06) when compared with the control group (0.74±0.05) (P < 0.01). In the MCF-7 cells, while it was observed that adhesion increased in the 10nM estrogen group (1.18±0.58) when compared to the control (0.69±0.17) and ethanol (0.66±0.12) groups, no statistically significant difference was found between the groups (P >0.05). In the MDA-MB–231 cells, it was observed that adhesion decreased in the 1nM estrogen group (0.69±0.05) when compared to the control (0.74±0.06) and ethanol (0.70±0.03) groups, but no statistically significant difference was found between the groups  (P >0.05piR-651 were observed in the 1nM androgen group (0.015±0.0002) when compared with the control group (0.003±0.0002) for the LNCaP cells  when compared with the control group (0.005±0.0002) for the LNCaP cells (piR-823 expression in the 10nM estrogen group (0.27±0.0002) when compared with the control group (0.54±0.0002) for the MCF–7 cells  method. The study by Yang et al. showed that miRNAs and piRNAs act as regulators in spermatogenesis, and it is a useful source for studies in this field . In theipiR-01551 and the long non-coding RNA site lnc00964-3 had a high risk of developing colorectal cancer [piR-Hep1 increased by 46.6% in hepatocellular carcinoma tissue when compared with healthy tissue. In the same study, it was argued that silencing of the gene site piR-Hep1 prevented the cancer cells from proliferating, migrating, and attacking the healthy tissue [In their study, Chu H. et al. investigated the single nucleotide polymorphism (SNP) of the sites containing piRNA clusters in the development of colorectal cancer. They determined that individuals that have the polymorphism of l cancer . The resy tissue . Anothery tissue . Hashim y tissue .piR-651 and piR-823 that we studied take part in the development of various cancers, especially gastric cancer [piR-823 in the tissue samples and cell lines of patients with multiple myeloma, and it was also observed that silencing of piR-823 in multiple myeloma cell lines resulted in dysfunction of the elements playing roles in the expression of protein associated with apoptosis and in the regulation of the cell cycle. Additionally, it was determined that piR–823 was directly associated with DNMT3A and 3B de novo DNA methyltransferases [piR-823 in the therapeutic xenograft models effectively inhibited tumor growth [piR-823 expression decreased in gastric cancer and displayed tumor suppressing properties both in vivo and in vitro [piR-823 expression that displays different properties  in different cancer types is thought to play a dual role in tumor formation. The data we obtained shows that androgen increases the piR-823 expression in LNCaP and PC-3 prostate cancer cells. The treatment of MDA-MB–231 cells with estrogen increases the piR–823 expression. Increasing the hormone levels in cancer cells by external administration of the hormones into these cells revealed that the piR-823 expression displayed oncogenic properties in prostate cancer cell lines and malignant estrogen-independent breast cancer lines, and that this property increased with the increase in hormone levels. The treatment of MCF–7 breast cancer cells with estrogen reduces the piR–823 expression. This result shows that piR-823 may have a different kind of expression in benign estrogen-dependent breast cancer cells treated with hormones. It was observed that therapy with the external administration of estrogen hormone had a positive effect in benign estrogen-dependent MCF-7 cancer cells and caused a decrease in the normally high piR-823 expression.It is postulated that the c cancer ,25,27,66sferases . As withr growth . AnotherpiR-651 cancer development. Previous studies showed that the piR-651 expression increased in various tumors, including gastric, colon, lung and breast tumors, when compared to healthy tissues [piR-651 and PIWI protein during cancer development may play a role in a variety of cancer types, including gastric, pancreas and oesophageal cancer [piR–651 may play a part in the metastasis of gastric tumor cells to the lymph nodes [piR-651 was silenced in gastric cancer [piR-651 expression increased in mesothelioma, hepatocellular, and cervical cancer [In our study, the piRNA site is thought to be the one that plays a role in  tissues . Additiol cancer –70. A reph nodes . It was c cancer . These sc cancer . Moreovel cancer .piR-651 and piR-823 increased upon administration, only the piR-823 expression was detected to have decreased in the estrogen-dependent breast cancer cell line MCF-7.Upon the determination of the concentrations at which the externally administered hormones increased cell proliferation the most, piRNA expressions were evaluated in these cells. It was observed that the viability was also at its highest level at the highest proliferation concentrations of the externally administered hormones while adhesion was reduced. On the contrary, a decrease in adhesion was detected in MDA-MB-231 cells. While it was observed that the expression of piR-651 expression increased in LNCaP and PC-3 prostate cancer cells treated with androgen and MCF-7 and MDA-MB-231 breast cancer cells treated with estrogen. The results of this study show that the increase in the expression of piR-651 and piR-823 during gonad development and in cancer may be associated with hormone levels in some cancer cells, and that these piRNA expressions vary depending on the hormone level and cancer cell type in the microenvironment of the cancer. We think that our study will contribute greatly to future studies on piRNAs and may be a source for the consideration of these properties in hormone therapies for some cancer types. More molecular studies are needed in order to obtain detailed information about certain results for the mechanisms of piRNAs in cancer.This study showed that piRNAs, which are becoming increasingly important today, may be affected by hormones. In general, it was observed that cell proliferation and viability increased with androgen or estrogen treatment and that cell adhesion decreased in cells other than PC-3 cells. It was detected that the S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(PDF)Click here for additional data file.S9 File(PDF)Click here for additional data file.S10 File(PDF)Click here for additional data file.


Endometrial cancer is the most common malignancy of the female genital tract worldwide, and endometrial endometrioid adenocarcinoma (EEC) is the major histological type of endometrial cancer. There is a great need for better markers with high sensitivity and specificity to permit early diagnosis and proper management of EEC. The aim of our study is to identify a miRNA classifier within plasma as a noninvasive biomarker for EEC diagnosis.This study was a retrospective case-control analysis which contained two independent cohorts including 93 participants. First, we screened 375 miRNAs in 29 plasma samples. 9 of the miRNAs were selected to be evaluated their expression by quantitative reverse-transcriptase polymerase chain reaction. A stepwise logistic regression model was then used to establish a new classifier in the validation cohort. Area under the receiver operating characteristic curve was used to evaluate the diagnostic accuracy. Co-expression analysis was used to verify the independence of results.miR-15b, -27a, and -223 were found to be differentially expressed in the EEC plasma between the two cohorts and had few connections with other miRNAs. The areas under the curve (AUC) were 0.768, 0.813, and 0.768 for miR-15b, -27a, and 223, respectively. miR-27a and CA125 can be combined as a potential non-invasive biomarker for detecting EEC, with the AUC of 0.894.Our study demonstrated three miRNAs, including miR-15b, -27a, and -233 have a good clinical value in EEC diagnosis. The classifier, including miR-27a and CA125, demonstrated a high accuracy in the diagnosis of EEC and might serve as a novel non-invasive biomarker in the future. Endometrial cancer is the most common malignancy of the female genital tract worldwide, and endometrial endometrioid adenocarcinoma (EEC) is the major histological type of endometrial cancer Just one decade ago, a novel class of evolutionarily conserved small (18–24 nucleotides) non-coding RNA, or microRNAs (miRNAs) was discovered as important regulators of gene expression Our study performed genome-wide miRNA expression profiles (375 miRNAs) from plasma samples of EEC patients and normal controls, and the validation was conducted in an independent cohort including EEC patients, patients with other endometrial diseases , and normal controls. We demonstrated miR-15b,-27a, and -233 has a great clinical value in diagnosing EEC, and a panel, which was combined with miR-27a and CA125, has a considerable clinical value in diagnosing EEC.Our study was approved by the ethics committee , and written informed consent was obtained from all study participants. The present study was divided into two phases: (1) Discovery phase. In this phase, global miRNAs profiling were screened in 29 samples using quantitative real-time PCR reaction. A Mann-Whitney test was used to discover differentially expressed miRNAs. (2) Validation phase. Nine candidates discovered were further validated in plasma samples of 64 participants by quantitiative RT-PCR (qRT-PCR). Three of them, which were found to be differentially expressed, were evaluate the diagnostic performance by area under the ROC (receiver operating characteristic) curve (AUC). (3) All the data of the participants in the validation phase were used to construct the diagnostic model by logistic regression.As shown in For plasma preparation, the whole blood (2 ml) was collected into EDTA tubes. Within 1 h at 4°C, the tubes were subjected to centrifuge at 820 g for 10 min at 4°C. The resulting plasma was transferred into new clear tubes, followed by a 10-min high-speed centrifugation at 16,000 g at 4°C to pellet the remaining cellular debris. Supernatant was transferred to clear tubes and stored at −80°C.−3 pmol) was added into the samples as the spike-in controls. The concentration was quantified by NanoDrop 1000 Spectrophotometer . The concentration of miRNA extrated from plasma ranged from 10 to 20 ng/µL.We extracted miRNA from 500 µL plasma by using miRcute miRNA Isolation Kit according to the instruction from the manufacturer . After the lysate was mixed into the plasma, cel-miR-39  polymerase in a 20-µL volume, and 6 µL of the poly (A) reaction solution was reverse transcribed to cDNA in another 20-µL volume using the Sharpvue miRNA First Strand Kit  to generate the first-strand cDNA. The reaction solution including cDNA products was diluted to one tenth with RNase free water and stored at −20°C before downstream quantitative PCR analysis. The single tube miRNA assays were used to detect and quantify mature miRNAs by Sharpvue 2× Universal qPCR Master Mix High Rox kit  and Sharpvue Human miRNA Primer Array kit  per the instructions in the kits. Each sample was detected by 375 miRNA primers and one spike-in miRNA (cel-miR-39) primers. miRNAs expression levels were quantified using ABI 7900HT Fast Real-Time PCR System . The reactions were incubated in a 384-well optical plate at 95°C for 2 min, followed by 3 cycles of 96°C for 5 s and 60°C for 1 min, 37 cycles of 96°C for 5 s and 60°C for 30 s, and running melting curve at the end. SYBR green dye and Rox dye were used as reporter and reference, respectively. The relative expression of each miRNA was calculated from the equation 2Data were analyzed using SPSS (version 19.0). For the data obtained by qRT-PCR, the nonparametric Mann-Whitney test was used for the comparison between EEC and control. A stepwise logistic regression was used to establish a model as a surrogate marker to diagnose the EEC. AUC was used as an accuracy index for evaluating the diagnostic performance of the significant miRNAs.The miRNAs were divided into two groups, and the Pearson correlation coefficients of the miRNA expression were calculated between each pairs. The co-expression miRNA pair was defined as correlation coefficient larger than 0.75 and P<0.05 −7), while another five miRNAs, including miR-1179, -4638-5p, -4665-5p, -3145-5p, and -4502 were significantly downregulated in EEC group , 0.813 , and 0.768  for miR-15b, 27a, and 223, respectively, while only 0.739  for CA125 .CA125+23.931×miR-27a-3.308. The ROC curve of the panel has an AUC of 0.894   was applied on the validation data set. miR-27a and CA125 could be combined as a potential classifier for detecting EEC, and the formula of the classifier was as follow: 0.097×= 0.970) .We generated a top500 co-expression miRNA network for cancer  and normEndometrial cancer is the most common malignancy of the female genital tract worldwide, and EEC is the major histological type of endometrial cancer. Currently the noninvasive diagnosis of EEC mainly relies on the combination of ultrasound, MRI, and serological markers, but none of them are completely satisfactory. CA125 has been used for many years as a serum marker for endometrial cancer diagnosis and screening. However, it has been recognized that CA125 levels has poor specificity in the detection of endometrial cancer. Other gynecologic malignancies such as ovarian cancer can also show the raise of CA125. There is a great need for better markers with high sensitivity and specificity to permit early diagnosis and proper management of EEC.A large number of miRNAs are proved to be present in circulation. They exhibit different profiles in patients with diverse diseases, including acute myocardial infarction It has been found that the circulating miRNAs has the capability to diagnose EEC according to the previous studies. Jia W. and his colleagues found a four-miRNA signature, including miR-222, -23, -186, and -204), might serve as a non-invasive approach for EEC diagnosis miRNAs in the circulation are extremely stable and protected from degradation by RNase activity or harsh environmental factors by microparticles Compared with other studies of circulating miRNAs in diagnosing EEC, our study is unique as the following reasons. First of all, we used not only healthy samples as controls, but also other endometrial diseases  into the research to avoid the risk of indistinguishable of EEC from other endometrial diseases. Secondly, considering there’s no standard endogenous control for the circulating miRNA studies, we used the spike-in control (cel-miR-39) in the study, which leaded to a highly reliable conclusion. Last but not least, one particular miRNA in circulation may help to distinguish between patients and controls, but lots of the researches would choose a panel of miRNAs to raise the sensitivity and specificity, which make the experimental result hard to be applied in clinical circumstance. Our study found that the combination of miR-27a and CA125 demonstrated high accuracy in diagnosis of EEC with an AUC of 0.894. This result includes only one new index; therefore reduces the test cost effectively.Although the miRNA classifier had a high diagnostic value in our research, there are still some limitations. First of all, as the difficulty in sample collection, the number of patients with atypical hyperplastic endometrium prevented us from testing in a more detailed classification. Studies including larger sample size are needed in the future. Secondly, considering the limited number of studies and the high cost of assay, it is still hard to extend the miRNA as a routine diagnostic biomarker into the clinical application. More researches and efforts are needed to change the experimental findings into clinical applications.In summary, our study demonstrated three miRNAs, including miR-15b, -27a, and -233 has a great clinical value in diagnosing EEC. The classifier, including miR-27a and CA125, demonstrated high accuracy in the diagnosis of EEC and might serve as a novel non-invasive biomarker in the future.


Cynosurus echinatus) is an annual grass, native to Europe, but also widely distributed in North and South America, South Africa, and Australia. Two hedgehog dogtail biotypes, one diclofop-methyl (DM)-resistant and one DM-susceptible were studied in detail for experimental dose-response resistance mechanisms. Herbicide rates that inhibited shoot growth by 50% (GR50) were determined for DM, being the resistance factor (GR50R/GR50S) of 43.81. When amitrole (Cyt. P450 inhibitor) was applied before treatment with DM, the R biotype growth was significantly inhibited (GR50 of 1019.9 g ai ha-1) compared with the GR50 (1484.6 g ai ha-1) found for the R biotype without pretreatment with amitrole. However, GR50 values for S biotype do not vary with or without amitrole pretreatment. Dose-response experiments carried out to evaluate cross-resistance, showed resistance to aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ) inhibiting herbicides. Both R and S biotypes had a similar 14C-DM uptake and translocation. The herbicide was poorly distributed among leaves, the rest of the shoot and roots with unappreciable acropetal and/or basipetal DM translocation at 96 h after treatment (HAT). The metabolism of 14C-DM, D-acid and D-conjugate metabolites were identified by thin-layer chromatography. The results showed that DM resistance in C. echinatus is likely due to enhanced herbicide metabolism, involving Cyt. P450 as was demonstrated by indirect assays (amitrole pretreatment). The ACCase in vitro assays showed that the target site was very sensitive to APP, CHD and PPZ herbicides in the C. echinatus S biotype, while the R biotype was insensitive to the previously mentioned herbicides. DNA sequencing studies confirmed that C. echinatus cross-resistance to ACCase inhibitors has been conferred by specific ACCase double point mutations Ile-2041-Asn and Cys-2088-Arg.Hedgehog dogtail ( Cynosurus echinatus) is an annual grass that is native to Europe, but is also widely distributed in North and South America, as well as South Africa and Australia. Plants are sensitive to herbicides that selectively inhibit the plastid enzyme ACCase, including diclofop-methyl (DM). Following many years of effective control, some reports have surfaced recently where plants survived lethal doses of DM. A hedgehog dogtail population in Chile was confirmed resistant to DM in 1999. In addition to exhibiting high levels of resistance to DM, cross-resistance to other ACCase inhibitors was also documented. The underlying basis for resistance has been ascribed to two separate mechanisms: enhanced metabolism of DM resulting from elevated activity of Cytochrome P450 monooxygenase; and a double mutation in a target site region of ACCase, resulting in Ile-2041-Asn and Cys-2088-Arg amino acid substitutions.Hedgehog dogtail (Cynosurus echinatus L.) is an annual grass, native to Europe, widely distributed in North and South America, South Africa, and Australia  may occur.Graminicide herbicides inhibit the chloroplastic acetyl coenzyme A carboxylase  action in the Poacea family, preventing fatty acid synthesis and reducing the production of the phospholipids used in the membranes . ChlorosC. echinatus biotypes to DM, and to other ACCase inhibitors , and to elucidate the mechanisms behind their resistance from foliar penetration, metabolic, ACCase assays and molecular basis for resistance to cross-resistance inhibitors in this biotype.The objectives of this research were to determine the resistance patterns of 14C]-diclofop-methyl  was provided by Bayer CropScience (Germany).. The clarified supernatant was applied to a desalting column previously equilibrated with S400 buffer. The ACCase enzyme was eluted from the column in 2 mL S400 buffer.The ACCase enzyme was partially isolated according to 14C]O3 into an acid-stable [14C]-product. The reaction product has been previously shown to be [14C]-malonyl-CoA , 50 μl enzyme fraction, and 5 mM acetyl-CoA in a final volume of 0.2 mL. Activity was assayed for 5 min at 34°C, and the reaction was stopped by adding 30 μl of 4 N HCl. A piece of filter paper was added to the reaction vial and the sample was dried at 40°C under a stream of air. After the sample was dried, ethanol-water  was added to the vial, followed by the addition of 5 ml of scintillation cocktail. Radioactivity was determined by LSS. Background radioactivity, measured as acid-stable counts (kBq) in the absence of acetyl-CoA, was subtracted from each treatment. One unit of ACCase activity was defined as 1 μmol malonyl CoA formed min-1. ACCase inhibiting herbicide concentrations resulting in a 50% inhibition of enzyme activity (I50) were determined in crude extracts. Data were pooled and fitted to the log-logistic model described previously. Experiments were repeated twice with five replicates per biotype.The enzyme activity was assayed by measuring the ATP-dependent incorporation of NaH[onyl-CoA . Assays C. echinatus R and S plants at the 2–3 leaf stage as in the penetration studies. A labeled herbicide was applied to the adaxial surface of the second leaf in 10 0.5 μL droplets by using a microapplicator. A total of 5000 Bq was used on each plant. Plants of the R and S biotypes were sampled 12, 24, 48, 72, and 96 h after treatment (HAT). Treated leaves were washed as described above. An aliquot of leaf wash solution was assayed for radioactivity, and the remaining solution stored at -20°C until analysis. Treated plants were split into shoots and roots were discarded. The shoots from each plant were powdered in liquid nitrogen by using a mortar and pestle. The powder was extracted with 4 mL of 80% methanol at 4°C and the homogenate centrifuged at 20 000 g for 20 min. The resulting pellet was washed with 80% methanol until no further 14C was extracted. The pellets were oven-dried and combusted as described above. The supernatants were combined, evaporated to dryness at 40°C under a stream of N2 at 10 kPa and re-dissolved in 500 μL of 80% methanol. DM and its metabolites in the supernatant were identified by thin-layer chromatography on 20 cm × 20 cm, 250 μm silica gel plates , using a 150/7/7 v/v/v toluene/ethanol/acetic acid mixture as mobile phase. Radioactive zones were detected with a radiochromatogram scanner (Berthold LB 2821) and their chemical nature was identified by comparing their Rf values with those for standards . The experiment was arranged in a completely randomized design with five replications per biotype.Herbicide metabolism was studied in leaf tissue from C. echinatus were sprayed with amitrole at 200 g ai ha-1 as explained in growth assays. After 8 h of applying amitrole, the DM metabolism was performed following the method previously proposed at 12, 24, 48, 72, and 96 HAT.At 3–4 leaf stage, R and S biotypes of -1. Foliar tissue (50 mg) of individual plants from the resistant and susceptible biotypes was taken before treatment for use in the molecular analysis. This rate eliminated all susceptible population plants 21 DAT , following manufacturer’s instructions, and DNA amount was quantified in a NANODROP Thermoscientific spectrophotometer . Each DNA sample was diluted until a final concentration of 10 ng/μl, which was immediately used for the polymerase chain reaction (PCR) or stored at -20°C until use.Table 1). Two sets of primers covering all five known mutation sites in region A  and B  (primers AC2F/R), were designed based on the chloroplastic ACCase sequences of other grass weeds, Alopecurus myosuroides (accession no. AJ310767), Bromus diandrus (AJ966446), Hordeum vulgare (AJ966456), L. multiflorum (AY710293), L. rigidum (AY995232), Zea mays (U19183). Ten individual plants from each biotype were genotyped.Two pairs of primers were designed to amplify regions in the CT domain known to be involved in sensitivity to ACCase herbicides (Thermus aquaticus (Taq) DNA polymerase. The PCR amplifications were performed with the following profile: 1 cycle of 94°C 2 min; 35 cycles of 30 s at 94°C, 30 seg at 58°C (for AC1781F/R primers) or at 60°C (for AC2F/R primers), and 1 min at 72°C; followed by a final extension cycle of 5 min at 72°C.ACCase gene fragments were amplified in a thermal cycler, with a final volume of 20 μl, containing 10 ng of template DNA, 0.75 μM of each primer, 1.6 μM of dNTPs, 2 μl of PCR buffer and five units of Amplified DNA fragments were purified using the Speed tools PCR Clean-Up Kit . Sequencing of the purified genomic DNA was done in the Genomic Unit Investigation Central Service of Badajoz University, Spain.50 values and the R/S ratios of both C. echinatus biotypes in response to several ACCase inhibitor herbicides are shown in Table 2. The resistance to DM was confirmed in the R biotype (GR50 = 1484.6 g ai ha-1) compared with the values found by the S one (GR50 = 33.9 g ai ha-1). The DM reduced the fresh weight of S biotype at low doses and eliminated plants at up to a dose of 500 g ai ha-1, whereas in the R biotype the same dose had no effect on the fresh weight and plants just died at up to a dose of 4000 g ai ha-1. The APP presented the highest FR values , while the R biotype has a high resistance to PPZ (pinoxaden), the cross-resistance corresponding to CHD (cycloxydim > setoxydim) herbicides being low (Table 2).The calculated GRC. echinatus biotypes . The pretreatment with amitrole significantly inhibited the growth of R biotypes (GR50 of 1019.9 g ai ha-1) compared with the GR50 (1484.6 g ai ha-1) found for the R biotype without pretreatment with amitrole. However, GR50 values for the S biotype did not vary and this was independent of amitrole treatment . The RF values were higher in plants (R) without amitrole (RF = 43.8) compared with those found in plants (R) pretreated with amitrole (RF = 31.4), indicating that Cyt. P450 was involved in DM-resistance in C. echinatus. However, reducing the resistance level by 28.3% cannot be explained only by the metabolism of the herbicide to non-toxic forms and (an)other additional mechanism(s) should be studied.The growth response to DM showed marked differences between 14C-DM increased with time in both C. echinatus R and S biotypes (Table 3). The 14C-DM peak foliar uptake occurred at 72 and 96 HAT for R and S plants. However, the S biotype seemed to reach the maximum peak penetration faster than the R biotype. Taken as a whole, foliar absorption levels did not seem to be involved in resistance between biotypes. Similarly, 14C-DM uptake and translocation were confirmed through phosphorimaging in both R and S biotypes . The herbicide was poorly distributed among leaves, roots and the rest of the shoot with unappreciable acropetal and/or basipetal DM translocation at 96 HAT. The picture taken through the phosphorimaging could not detect the difference in DM translocation from the treated leaf to the root in either biotype of C. echinatus found in the above test . These results suggest that an altered symplast transport is not responsible as a mechanism for DM-resistance in C. echinatus.In general, more than 93.0 ± 2.5% of the total applied radioactivity was recovered in the uptake, translocation, and metabolism study. Foliar uptake of C. echinatus R and S biotypes . However, DM was de-esterified to D-acid significantly faster and greater in the S than in the R biotype, while D-acid remained nearly constant (80%) in S biotype over the time (from 12 to 96 HAT), although in the R biotype it dropped to below 8.8% at 96 HAT . This is because in the R biotype, monooxygenases Cyt. P450 quickly formed a D-hydroxylated metabolite and then a nontoxic D-conjugate. Although this route was also followed by the S biotype, the formation speed of the D-conjugate was slower (0.24) than of the R biotype .Metabolites  were found in both Figures 3C,D), but not in S biotypes, where the polar D-conjugate went on being minimum .Foliar pretreatment with amitrole significantly inhibited DM metabolism to the polar D-conjugate in R biotypes . Based on the obtained resistance factors (FR), it was clear that there was a cross- resistance to the three ACCase inhibitor herbicide groups studied (Table 4). Evidently, the R biotype of C. echinatus was highly resistant to pinoxaden (PPZ), moderately resistant to fenoxaprop, diclofop, and cyhalofop (APP) and poorly resistant to setoxydim and cycloxidim (CHD) herbicides. The results suggest a less sensitive form of ACCase in the R biotype than in the susceptible biotype, determining this characteristic cross-resistance to ACCase inhibiting herbicides. The resistance in the C. echinatus R has been related to a mutation in the ACCAse gene diminishing the target site sensitivity to the herbicides.The ACCase C. echinatus showed six non-synonymous SNP’s respect to A. myosuroides corresponding to the 1789, 2041, 2065, 2067, 2077, and 2088 positions (Table 5). The 1789, 2065, 2067, and 2077 positions were considered unrelated to resistance. The sequences obtained of C. echinatus were aligned to each other and with the CT domain of plastidic ACCase genes of other grass weeds . The nucleotide sequences of the A region between the R and S biotype did not differ. In the B region, the R biotype showed two SNP’s compared to S biotype resulting in a double substitution in the target-site. A codon change from ATT to AAT resulted in an amino acid substitution of Ile-2041-Asn, whereas, a codon change from TGT to CGT resulted in an amino acid substitution of Cys-2088-Arg in the R biotype (Table 5). No other mutations were found (data not shown).Two fragments of 229 and 478 bp of the CT domain of the ACCase gene corresponding to the A and B region, respectively, were sequenced. Single nucleotide polymorphisms (SNPs) were found in the coding sequence. 450 , this compound being more polar than D-acid and not at all phytotoxic (450 inhibitors (amitrole and ABT), the amount of nontoxic D-conjugates formed 96 HAT in R biotypes was reduced by over 10 times the amount of D-conjugate metabolites observed in R biotypes not preincubated with amitrole  in R and S biotypes was found when addressing the recovered radioactivity. Over time, small differences in the translocation from the treated leaf to the roots were detected in the S biotype with respect to the R one. However, these results did not imply considering uptake and translocation as a determining DM-resistance mechanism mainly due to the large amount of herbicide remaining in the treated leaf (Table 3). These results are in agreement with others obtained in our laboratory . Previous cases of APP and CHD herbicide resistance in grasses correlating with reduced sensitivity in the target enzyme have been reported. Target site resistance is essentially caused by a single amino acid change in the CT domain, which impacts the effective binding of ACCase-inhibiting. Seven different codons  responsible for resistance have been described in grassy weeds  at the CT domain of the ACCase gene. As a consequence this herbicide group is becoming ineffective in C. echinatus control and farmers and technicians must be very careful with the use of selective alternative herbicides with different action mechanisms because Cyt. P450 monooxygenase is unpredictable and can trigger herbicide resistance with similar mechanisms or even different action mechanisms, including herbicides never used. Farmers should consider the integration of a pasture phase, which allows the use of grazing and the use of non-selective herbicides in order to prevent the weed seed productions. An integrated weed management (IWM) is required to allow the reduction and elimination of herbicide-resistant handling populations.This study is the first documented case where two mechanisms (RST and NRST) are involved in cross-resistance to ACCase inhibiting herbicides in hedgehog dogtail collected in Chile. The resistance is associated with an enhanced metabolism mediated by Cyt. P14C-DM absorption/translocation, and 14C-DM metabolism; RA, and MO performed the ACCase gene sequencing.PF, and HC-H performed the DM plant response and ACCase activity assays; RA, PF, and RD performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By using one stage amplifier in the anomalous dispersion regime, the laser can be amplified up to 450 mW and the narrowest pulse duration of 1.4 ps can be obtained simultaneously. Our work paves the way to graphene Tm/Ho co-doped mode-locked all-fiber master oscillator power amplifiers as potentially efficient and economic laser sources for high-power laser applications, such as special material processing and nonlinear optical studies.Mid-infrared ultrafast fiber lasers are valuable for various applications, including chemical and biomedical sensing, material processing and military applications. Here, we report all-fiber high-power graphene mode-locked Tm/Ho co-doped fiber laser at long wavelength with evanescent field interaction. Ultrafast pulses up to 7.8 MHz are generated at a center wavelength of 1879.4 nm, with a pulse width of 4.7 ps. A graphene absorber integrated with a side-polished fiber can increase the damage threshold significantly. Harmonics mode-locking can be obtained till to the 21 Long-wavelength >1.55 μm) ultrafast lasers can potentially be applied in various applications, such as free-space optical communication due to atmospheric transparency window, molecular spectroscopy, medical diagnostics, laser surgery, long-range radar, and gas detection.55 μm ul23Mode locking is the most typical approach to generate ultrashort pulses. Various methods have been reported to achieve mode locking: Nonlinear polarization rotation (NPR) is the one of the most commonly used methods for fiber laser mode locking, which have been studied for many years6791112131415171820192527In order to generate ultrashort pulse at wavelength above 1.55 μm and even up to 3.9 μm3234353637In this paper, we utilize the side-polished fiber together with graphene grown by CVD method to achieve mode locking in a Tm/Ho co-doped ring fiber laser. It is demonstrated that mode locking can be obtained at a center wavelength of 1879.4 nm, spectral bandwidth of 1.8 nm, and pulse width of about 4.7 ps. High-order harmonic mode locking (up to 164.6 MHz) can be obtained with the increase of pump power. After we use a Tm-doped fiber amplifier (TDFA) to boost the output power, the maximum output power is ~450 mW, corresponding to the peak power of about 9.7 kW. Our results shows graphene all-fiber Tm/Ho co-doped mode-locked fiber laser with single mode output and high peak power. These unique properties enable the advantages of compactness, good beam quality and high power output, which make them potential candidates in the molecular spectroscopy, nonlinear frequency conversion, and laser surgery applications.2, Graphene Supermarket) is transferred onto a side-polished fiber embedded in a glass plate by a modified transfer process. W~996000 by GPC, Sigma Aldrich, dissolved in chlorobenzene with a concentration of 50 mg/mL), which is then cured at 110 °C for 10 min. The underneath Cu foil is then dissolved in an aqueous (NH4)2S2O3 solution (10%). The remaining PMMA/graphene film is cleaned by repeatedly dipping it in deionized water, and then a 0.5 M HCl bath for 5–10 min. Then, the PMMA/graphene film is picked up through the target side-polished fiber. After drying in the ambient condition, some PMMA solution is dropped onto the side-polished fiber to dissolve the pre-coated PMMA and flatten out winkles on the graphene. The PMMA is then dissolved in acetone, leaving SLG on glass with side polished fiber.CVD grown single layer graphene (SLG) (~20 × 20 mmG/I2D) is 0.54, which is recognized as intrinsic single layer grapheneAfter transfer, the graphene on the side-polished fiber was characterized by Raman spectroscopy (under 532 nm excitation) and absorption microscopy. A representative Raman spectrum of the transferred graphene on side-polished fiber is shown in When the pump power reaches 371 mW, mode-locking can be self-started. When we decrease the pump power, pulse train can be maintained till 326 mW, which is due to the hysteresis effect2 profile with a pulse width of 4.67 ps. The corresponding 3-dB spectral width is 1.8 nm, corresponding to a time-bandwidth product (TBP) of about 0.714. The TBP is larger than transform-limited sech2 pulse, which indicates the pulses are chirped. Compared with Ref. 2, is comparable. The pulse width can be further reduced by controlling the dispersion of the cavity.The typical output of the fiber laser  is shown in th harmonics mode locking, which gives an output of 10.1 mW with a repetition rate of 164.6 MHz. Because of the limitation of the damage threshold of the fiber components (500 mW), the pump power are not further increased. th, 8th, 11th, 15th, and 21th harmonic mode locking versus the different pump power. With further increasing the pump power, our fiber laser can operate from the fundamental mode locking regime to the high-order harmonic mode-locking regime. th harmonic, which is limited by the damaged threshold of the WDM coupler rather than the absorber. The absorber with a sandwich structure, however, cannot bear such high pump power. Thus, it is difficult to observe higher-order harmonics mode locking based on the sandwich-structure absorber.The absorber structure in our work is quite different from those with the sandwich structure. In our work, higher-order harmonic mode locking is observed till to the 21To increase the output power of our graphene based fiber laser, we use a home-made TDFA to amplify the seed laser. The maximum output power after amplification is ~450 mW by removing the residual pump power, when the passively mode-locked fiber oscillator operates at the fundamental mode locking (output power of the oscillator is 3 mW). The amplification coefficient is more than 21 dB. The spectral evolution at different output power is shown in Conventionally, chirped pulse amplification (CPA) technique can overcome the pulse break-up, which is widely used in the normal-dispersion regime. After being amplified, the pulse can be further compressed due to the linear chirp of the pulse. Different from the CPA technique, the pulse can be amplified and compressed simultaneously in the anomalous dispersion regime. This can help to get shorter pulse than the transform-limited pulse due to the nonlinearity and high order soliton effect. Our approach provides a scheme to further compress the transform-limited pulse.In order to demonstrate that the mode locking is solely mode locked by the SLG SA with graphene, we remove the side polished fiber with graphene or just remove the graphene on the side-polished fiber. No clear mode-locked pulse train or typical mode-locked spectrum can be observed. Only the CW regime was obtained from the laser cavity, which confirms that the mode locking is solely contributed by the side-polished fiber with graphene. The stability of our laser has been tested for a few days, no performance degradation was observed.th order, which is limited by the damaged threshold of fiber components. The output power of the proposed fiber laser can be further amplified to more than 450 mW, corresponding to a peak power of about 9.7 kW. The high peak-power Tm/Ho co-doped mode-locked fiber laser, with single mode output and high beam quality, can be potentially applied with further development in applications such as laser surgery, medical diagnostics, and gas detection, etc.In conclusion, we demonstrate passively mode-locked high power Tm/Ho Co-doped fiber laser based on a side-polished fiber incorporated with CVD grown graphene. The obtained pulse width is about 4.7 ps, the spectral width is about 1.8 nm with Kerr side band, indicating that the fiber laser operates in the conventional soliton regimes. With further increase of the pump power, the Tm/Ho co-doped mode-locked fiber laser can operate in high-order harmonic mode locked states till to the 21Output spectra of our fiber lasers are measured through an optical spectrum analyzer . Mode-locked pulse train and radio frequency (RF) spectra are detected by using a 33-GHz mixed signal oscilloscope , one signal source analyzer  coupled with 12-GHz photodetector (Newport 818-BB-51F), respectively. A wide-range autocorrelator (From Femtochrome FR-103HP) is used to detect output pulse width.How to cite this article: Li, X. et al. High-power graphene mode-locked Tm/Ho co-doped fiber laser with evanescent field interaction. Sci. Rep.5, 16624; doi: 10.1038/srep16624 (2015).


Postoperatively, diffuse blebs with ridges were found in all eyes in group A, two blebs were observed in group B, and no bleb formation was present in group C. The postoperative central anterior chamber depth in group A was significantly less than that of the other two groups. The postoperative intraocular pressure of group A stabilized at 6.33–8.67 mmHg, whereas that of group C gradually remained at 7.55–10.02 mmHg. The histopathology showed that the fibrous tissue thickness of the blebs in group A was significantly thinner than that of the other groups. We conclude that the 5-Fu-polycaprolactone sustained-release film had a sustained drug release effect, which promoted the inhibition of bleb scarring after Ahmed glaucoma valve implantation.This study was designed to investigate whether 5-fluorouracil (5-Fu)-polycaprolactone sustained-release film in Ahmed glaucoma valve implantation inhibits postoperative bleb scarring in rabbit eyes. Eighteen New Zealand white rabbits were randomly divided into three groups . Group A received combined 5-Fu-polycaprolactone sustained-release film application and Ahmed glaucoma valve implantation, group B received local infiltration of 5-Fu and Ahmed glaucoma valve implantation, and group C received Ahmed glaucoma valve implantation. Postoperative observations were made of the anterior segment, intraocular pressure, central anterior chamber depth, blebs, drainage tube, and accompanying ciliary body detachment. The pathology of the blebs and surrounding tissues were observed at month 3 postoperatively. We revealed that the 5-Fu-polycaprolactone sustained-release film maintained a release concentration range of 13.7 ± 0.12 to 37.41 ± 0.47 μg/ml over three months  Ahmed glaucoma valve (AGV), which was granted clearance for sale by the Food and Drug Administration (FDA) in 1993, has become the preferred treatment for refractory glaucoma. Clinical studies have confirmed that compared with other aqueous humor drainage devices, the AGV (FP7/8) has a relatively lower incidence of postoperative complications . The sucPolycaprolactone  is a novel biodegradable polymer material, which has been prepared as sustained-release microspheres, intraocin vitro. We then investigated the in vivo inhibitory effects of 5-Fu-PCL sustained-release film on bleb scarring after AGV implantation in rabbit eyes.Taking advantage of PCL’s film-forming property, we prepared a 5-Fu-PCL sustained-release film using spray-forming methods, and observed the release effects of the film All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and protocols were approved by the Institutional Animal Care and Use Committee in Wenzhou Medical University. The animals were housed in an air-conditioned room with an ambient temperature of 16–26°C, a relative humidity of 40–70% and a 12-hour light-dark cycle with a daytime light intensity of approximately 500 lux. Rabbits were provided with a commercial primate diet. In addition, fresh vegetables were provided twice daily, and water was freely available at all times. All examinations were performed gently and all efforts were made to minimize suffering. Topical proparacaine HCl  was used before measuring IOP, AS-OCT and slit-lamp examination. All surgical manipulations were carried out under general anesthesia induced with an intramuscular injection of 0.25 ml/kg Sumianxin II  and 1 ml/kg of 3% sodium pentobarbital . The health of these animals was monitored every day after surgery through observing and analyzing the growth vigor, diet and activity. Tobradex ointment  was used three times for 7 days to alleviate suffering during post-surgery recovery. Animals were sacrificed with an intravenous injection of an overdose of sodium pentobarbital solution .5-Fu  was ultrasonically dispersed in 5 ml of acetonitrile, and PCL  was ultrasonically dissolved in 15 ml of dichloromethane. After mixing, the solution was sprayed using a LPH-50 spray gun  on a polytetrafluoroethylene plate with nitrogen stream at 0.2 MPa. After vacuum drying for 12 h, the resultant sustained-release film was cut using a mold to a diameter of 6 mm and a thickness of 0.35 ± 0.01 mm (mean ± standard deviation [SD]). The drug loading of the prepared 5-Fu-PCL sustained-release film was 4.13 ± 0.12 mg.in vitro drug release of the sustained-release film was determined by measuring the concentrations of 5-Fu in the different samples using high-performance liquid chromatography (HPLC) [As previously reported, three parallel samples of the 5-Fu-PCL sustained-release film were respectively placed in a dialysis bag  and put into a centrifuge tube filled with 10 ml phosphate buffered saline  [y (HPLC) . Each meEighteen healthy New Zealand white rabbits weighing 2.0–2.5 kg were randomly divided into three groups  using a random number table. In each animal, an operation was performed on the right eye. Group A received combined 5-Fu-PCL sustained-release film application and AGV implantation. Group B received local infiltration of 5-Fu and AGV implantation. Group C received AGV implantation. All animals were given adaptive feeding for 3 days. Binocular IOP was measured with a Tono-Pen tonometer . The depths of the anterior segment and the central anterior chamber, as well as the degree of ciliary body detachment were evaluated by anterior segment optical coherence tomography  .All experimental procedures were performed by the same surgeon (Weihua Pan). After sterilization with ethylene oxide, the 5-Fu-PCL sustained-release film was fixed with a 10–0 suture on the AGV . The ani®) eyedrop was given the operated eyes for one week after the operation.In group A, AGV with the 5-Fu-PCL sustained-release film was implanted in the scleral surface. In group B, after fixing an AGV on the scleral surface, a 5 mm × 3 mm piece of cotton soaked with 25 mg/ml 5-Fu (volume 140 μl) was placed on the surface for 5 minutes, and then the surgical field was rinsed with 100 ml saline. In group C, the eyes were simply implanted with AGV. In all experimental animals, the AGV was placed between the rectus and lateral rectus, 7 mm from the front edge of the limbus. It was sutured with 2 needles using 5–0 polyester line  and then fixed on the scleral surface. A tobramycin-dexamethasone combination  was performed to compare groups at each time point. The statistical analysis was performed using SPSS16.0 software. A in vitro drug release of the 5-Fu-PCL sustained-release film was maintained for at least 3 months. The regression analysis of the release pattern showed that the dissolution curve of the drug release was in accordance with the Higuchi equation: Y = 5.6758t1/2+9.6786, R2 = 0.9948. This result indicated that the 5-Fu-PCL sustained-release film had the features of a long-acting preparation. In addition, the in vitro drug release of the 5-Fu-PCL sustained-release film was relatively stable with a concentration range of 13.7 ± 0.12 μg/ml to 37.41 ± 0.47 μg/ml over 3 months as shown in The All animals survived from the surgical procedures. Postoperatively, no abnormalities of anterior and posterior segments were found in all animals. The location of the drainage tube in the anterior chamber was also normal, and the drainage was effective without leakage on the blebs in each animal. The blebs in group A were diffuse and had ridges .As shown in The preoperative IOP was 15.33 ± 0.97 mmHg in group A, 15.22 ± 1.16 mmHg in group B, and 16.00 ± 0.86 mmHg in group C. There were no significant differences among the three groups. By the 14th postoperative day, the IOP of group A had stabilized at 6.33–8.67 mmHg, whereas the IOP of group C had gradually increased to 7.55–10.02 mmHg. The difference in the IOP between groups A and C was statistically significant; however, the IOP in group B was not statistically different from those of the other groups .The preoperative central anterior chamber depth was 2.421 ± 0.018 mm in group A, 2.429 ± 0.026 mm in group B, and 2.392 ± 0.033 mm in group C. On the 3rd month postoperatively, the central anterior chamber depth was 1.960 ± 0.028 mm in group A, which was significantly different from that of group B (2.357 ± 0.040 mm) and group C (2.327 ± 0.051 mm) .In this study, we successfully prepared a 5-Fu-PCL sustained-release film using a spray-forming method. Along with having a simple preparation process, the 5-Fu-PCL sustained-release film has several other advantages, including sufficient drug volume, stable release, and long-duration drug delivery. Our animal experiments and pathological findings indicate that combined AGV implantation and 5-Fu-PCL sustained-release film application in rabbit eyes was safe, and this approach effectively inhibited bleb scarring after the AGV implantation.IOP depends on the encapsulation of the drain surrounding the rear disc, the resistance of the encapsulation wall, and the surface area of the encapsulation. If the encapsulation wall is thinner or if the surface area of the encapsulation is greater, then the IOP is lower. Lloyd ein vitro studies performed by Leonard et al. [in vitro. These results indicated that PCL film might have potential as a human conjunctival tissue engineering scaffold material. Another report by Armando et al. [The d et al.  demonstro et al.  confirmeIn comparison to local infiltration of liquid 5-Fu, a significant advantage of combined AGV and 5-Fu-PCL sustained-release film implantation is that it maintains functional blebs for at least three months. As confirmed by Cantor et al.[We sutured the drainage tube with one needle using an 8–0 absorbable suture to limit the drainage of the aqueous humor and to prevent the occurrence of continuously low IOP and a shallow anterior chamber, as reported in the literature . Based oWe found that the fibrous tissue surrounding the blebs was thicker than that reported by Nurettin et al. . We hypoEarly tissue repair after AGV implantation is an important factor affecting the success of the surgery. Bleb scarring after AGV implantation has been shown to be ongoing, and the one-year success rate after AGV implantation declines due to bleb scarring . In the One limitation of this study is the choice of experimental animal. We chose healthy New Zealand white rabbits as the experimental subjects. These animals had normal aqueous humor drainage pathways compared to the subject with glaucoma. Therefore, aside from drainage by the AGV, a portion of the aqueous humor could also be discharged through the normal channels for aqueous humor outflow after the operation. This condition differs slightly from the real glaucoma condition, in which the aqueous humor drainage pathways are defective to some extent. This fact can be inferred from the observation of the lower postoperative IOP of rabbits in the three groups compared to the preoperative values. Another limitation of this study is that we only observed postoperative bleb scarring for up to 3 months. The presence of persistent scarring after AGV implantation may require a longer term to observe the histopathological changes of the blebs.In summary, the 5-Fu-PCL sustained-release film used in the present study fulfills the requirements of sustained-release pharmaceutical preparations. In a rabbit eye model, our results demonstrate that implantation of the AGV with 5-Fu-PCL sustained-release film effectively inhibited bleb scarring.


Neutrophil elastase (NE) is an early myeloid-specific serine protease, which is predominantly produced by promyelocytes. A previous study demonstrated that NE has an important role in the development of acute promyelocytic leukemia (APL). The process of APL was shown to be accelerated in animals that expressed abundant NE, whereas NE-deficient mice were protected from APL development; thus suggesting an important role for NE in the development of APL. The present study aimed to investigate the effects and possible mechanisms of NE. Up- and downregulation of NE in various leukemia cell lines was conducted in order to explore its significance in the occurrence and procession of leukemia, with the aim of identifying novel targeted therapeutic drugs for the treatment of leukemia. NE was overexpressed in cells following infection with an adenovirus, and Cell Counting kit-8 and flow cytometry results demonstrated that cell proliferation was promoted, and cell apoptosis was inhibited, as compared with the untreated cells. NE was downregulated in the cells by both RNA interference and treatment with GW311616A, a specific inhibitor of NE, following which cell growth was shown to be inhibited and apoptosis was induced. These results suggested that NE may promote the development of APL, therefore, NE may be a therapeutic target and its inhibitor GW311616A may be a potential therapeutic drug for leukemia. Furthermore, the apoptosis-associated protein B-cell lymphoma 2 (Bcl-2)-associated X protein was significantly increased, whereas Bcl-2 was markedly decreased in the cells with downregulated NE. Further experiments revealed that the probable apoptosis-associated signaling pathway was the phosphoinositide 3-kinase/AKT pathway. The present study is the first, to the best of our knowledge, to demonstrate that GW311616A, a specific NE inhibitor, may act as a potential targeted drug for leukemia, which may have a profound impact on the future of leukemia-targeted therapy. Neutrophil elastase (NE) is a neutrophil-specific serine protease, which is able to degrade virulence factors and kill bacteria. NE knockout mice are susceptible to bacterial and fungal infections ,2. It haNE is predominantly produced in promyelocytes. Lane and Ley  demonstrNE inhibitors, both endogenous and synthetic, are able to inhibit NE activity and regulate the release of inflammatory cytokines and chemokines . EndogenIn our preliminary experiment, NE expression was detected in five cases of APL (unpublished data), and NE expression was higher in patients with APL, as compared with healthy controls, thus confirming its important role. In order to clarify the role of NE in the process and development of leukemia, two different leukemia cell lines were selected: K562 and U937. The K562 cells express NE at low levels, whereas U937 cells contain abundant NE ,9.To explore the possibility of NE as a target for diagnosis and treatment, NE was upregulated in K562 cells by recombinant adenovirus, and silenced in U937 cells by small interfering (si)RNA and treatment with a specific inhibitor of NE, GW311616A. The exact effects of NE were then determined on the various cell lines, including alterations in cell proliferation, apoptosis, expression of apoptotic proteins, and activation of related signaling pathways.2. The medium was refreshed daily. An Ad-Easy system  was used to construct the recombinant adenovirus  were cultured in RPMI 1640  supplemented with 10% fetal bovine serum (Gibco Life Technologies) at 37°C in an environment containing 5% CO5/ml) were cultured in 24-well plates, and were infected with 5 μl recombinant adenovirus Ad-NE (containing NE) and Ad-KZ (empty vector), conjugated to GFP. Fluorescence microscopy was used to observe green fluorescence 48 h post-infection. The cells were divided into three groups: Infection group (K562/Ad-NE), empty vector group (K562/Ad-KZ) and untreated K562 cell group.K562 cells (2×105 logarithmic growth phase cells) were seeded into 6-well plates. For cell transfection, 5 μl siRNA and 5 μl Lipofectamine® 2000  were diluted in 100 μl Opti-MEM (Invitrogen Life Technologies) separately. The siRNA and Lipofectamine® 2000 were then gently mixed and incubated for 25 min at room temperature. The siRNA-Lipofectamine® 2000 complexes were subsequently added to each well and mixed by gentle agitation. All siRNA were purchase from RiboBio  and the sequences of the NE-specific siRNAs were as follows: siRNA101, 5′-CCGUAAACUUGCUCAACGAdTdT-3′ and 3′- dTdTGGCAUUUGAACGAGUUGCU-5′; siRNA102, 5′-CCGGUGGCACAGUUUGUAAdTdT-3′ and 3′-dTdTGGCCACCGUGUCAAACAUU-5′; siRNA103, 5′-GAUCGACUCUAUCAUCCAAdTdT-3′ and 3′-dTdTCU-AGCUGAGAUAGUAGGUU-5′. Following a 48 h incubation at 37°C, red fluorescence protein (RFP) was visualized by fluorescence microscopy. Transfection efficiency was determined by calculating the number of cells expressing RFP. The cells were divided into the following experimental groups: Untreated group, negative control (NC) group, siRNA-101 group, siRNA-102 group and siRNA-103 group.Cell density was 60–80% confluent on the day of transfection. The U937 cells (5×104 cells/well), and 150 μmol/l GW311616A  was subsequently added to the cells. After 48 h, the protein was extracted using radioimmunoprecipitation lysis buffer  and concentration was measured by a bicinchoninic acid assay (BCA). Purified human leukocyte elastase (Sigma-Aldrich) was used as a standard, and was diluted in pure water to provide the following final concentrations: 100, 200, 400, 800, 1,600 and 3,200 pmol/l. Another three wells contained the protein samples. A total of 100 μl substrate solution (Sigma-Aldrich) was added to the samples and incubated at 37°C for 1 h. The optical density (OD) was measured at 405 nm in an ELISA analyzer. According to the OD value and a linear regression equation of the standards, the activity of NE in the samples was calculated.The U937 and K562 cells were seeded into a 6-well plate (6×106) were collected in Eppendorf tubes, washed with ice-cold phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation solution containing a protease inhibitor cocktail . Protein concentration was determined using the BCA method. A total of 30–50 μg protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was then transferred to a polyvinylidene difluoride membrane . The membrane was blocked with 5% skimmed milk (EMD Millipore) for 2 h at room temperature, and was then incubated with the following primary antibodies overnight at 4°C: Mouse anti-human NE monoclonal antibody ; and rabbit anti-human polyclonal B-cell lymphoma 2 (Bcl-2), rabbit anti-human polyclonal Bcl-2-associated X protein (Bax) and rabbit anti-human AKT polyclonal antibodies . The membranes were then incubated with goat anti-rabbit and goat anti-mouse secondary antibodies  for 1 h at 37°C. After washing with Tris-buffered saline containing Tween-20 (TBST), the immunoreactive complexes were visualized using an enhanced chemiluminescence system . β-actin was used as an internal positive control.The cells (5×103/well). A total of 10 μl CCK-8 was added to the cells, in order to quantify cell proliferation 1 to 4 days post-infection. Following a 2 h incubation, the absorbance of each well was measured at 450 nm, using a spectrophotometer . A cell growth curve was generated with absorbance as the ordinate and time as the abscissa. The experiment was repeated three times.The cells were plated into 96-well plates (5×10μl Annexin-V-fluorescein isothiocyanate fluorescent dye and 5 μl propidium iodide (Sigma-Aldrich). The rate of cell apoptosis was analyzed using a FACsorter  following a 15 min incubation at room temperature. Furthermore, 5×105 cells were collected, centrifuged at 500 × g for 5min and washed with pre-cooled PBS twice. The supernatant was discarded and precooled 70% ethanol (1 ml) was added and the samples were fixed overnight at 4°C. Following fixation, the samples were centrifuged at 500 × g for 5 min and washed with 1 ml PBS twice. The samples were resuspended cells in 100 μl 1 mg/ml RNase solution in a 37°C water bath for 30 min. Following incubation, 150 μl 50 μg/ml propidium iodide staining solution was added and incubated for 30 min in dark at room temperature. The cell cycle distribution was detected using a FACsorter. Experiments were repeated three times.The cells were routinely collected and centrifuged at 500 × g for 5 min at room temperature. After the cells had been washed twice with PBS, a staining mixture was prepared containing 5 Statistical analyses were performed using SPSS 17.0 software . Experimental results are presented as the mean ± standard deviation. An independent samples t-test was used to compare the results of two groups. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated at least three times.To upregulate NE gene expression, the K562 cells were infected with recombinant adenovirus Ad-NE or Ad-KZ with enhanced green fluorescent protein (GFP). After 48 h, the percentage of GFP-positive cells in the Ad-NE and Ad-KZ infection groups was 70 and 60%, respectively . These rin vitro, CCK-8 and flow cytometric analysis were performed to detect the proliferation and apoptosis of the K562 cells, respectively. The CCK-8 assay showed that cell growth was markedly enhanced in the Ad-NE group in a time-dependent manner, as compared with the untreated control and Ad-KZ groups (P<0.05, To determine whether upregulation of NE had an effect on cell growth and apoptosis  P<0.05, . Flow cy P<0.05, .In order to determine the function of NE in leukemia, the present study knocked down its endogenous expression by RNA interference. The percentage of RFP-positive cells in the U937/siRNA group was 85% 48 h post-transfection . The effIn order to accurately evaluate the effect of NE knockdown, the growth and apoptosis of siRNA-infected and GW311616A-treated U937 cells was detected. Following downregulation of NE expression, cell proliferation was inhibited in a time-dependent manner . Flow cyIn the GW311616A-treated group, the rate of apoptosis was enhanced, as compared with the control group P<0.05, .To further investigate the effects of NE on apoptosis in leukemia, the expression levels of relevant apoptosis-associated proteins Bax and Bcl-2 were detected following NE knockdown in U937 cells. Downregulation of NE with siRNA-103 was able to increase the protein expression levels of Bax and decrease the expression of Bcl-2, as compared with the control group . FollowiIn order to determine the underlying mechanism of the effects of siRNA and GW311616A on the proliferation and apoptosis of leukemia cells, the expression levels of AKT and phosphorylated-AKT in the PI3K/AKT signaling pathway were detected. The expression levels of phosphorylated AKT were decreased, whereas the expression levels of total AKT were similar in each group, which suggested that the siRNA inhibited the activation of the AKT signaling pathway .In recent years, numerous studies have focused on leukemia, particular aims have been exploration of its therapeutic targets and drug interventions, and identification of novel approaches for molecular diagnosis.et al (NE, encoded by Ela-2, is a type of serine protease , which iet al ,9, whoseIn order to explore the exact effects of NE and to clarify its underlying mechanism, the present study upregulated NE in K562 cells, which express little NE; and downregulated NE in U937 cells, which contain various levels of NE. Following up- or downregulation of NE, the proliferation and apoptosis of the two cell lines was measured, and the results demonstrated that NE had a proliferation-inducing and anti-apoptotic effect in leukemia cells. The cell cycle of the Ad-NE infected cells was arrested in S-phase, indicating that NE may be a potential therapeutic target.It is well-known that apoptosis is characterized by a series of biochemical events, including condensation of the nuclei, DNA fragmentation, and ultimately cell death ,22. The The results of the present study demonstrated that silencing NE by siRNA in U937 cells resulted in an upregulation of the expression of Bax, whereas Bcl-2 expression was downregulated. Further experiments demonstrated that these findings may be achieved through regulation of the PI3K/AKT pathway, which has an important role in orchestrating various cellular processes, including proliferation, differentiation and apoptosis –31. The To further understand the effects of NE, and to identify therapeutic targets for leukemia, a specific inhibitor of NE, GW311616A was selected. GW311616A is a potent, intracellular, orally bioavailable, and long-acting inhibitor of human NE ,32. In tIn the present study, the protein expression levels of apoptosis-inducing Bax were reduced and the expression levels of anti-apoptotic proteins were increased following downregulation of NE, by siRNA and treatment with GW311616A. These results indicated that NE may be a therapeutic target for leukemia, and the inhibitory drug GW311616A may be used as a potential treatment. However, it will be beneficial to continue studying the effects of NE inhibition in various leukemia cell lines, including HL60 cells.The present study demonstrated that upregulating NE was able to promote the growth of leukemia cells and decrease the proportion of apoptotic cells. Conversely, downregulation of NE by siRNA and GW311616A treatment inhibited proliferation and induced apoptosis in leukemia cells. This novel information regarding the involvement of NE in leukemia cell proliferation and apoptosis may be used to identify a potential biomarker or treatment target for leukemia, and the NE-specific inhibitor GW311616A may be a promising treatment target option.


N = 43) to train on either the n-back (including 2-back and 3-back levels) or an active control task. Following training, dMTS was administered in the fMRI scanner. The n-back group performed marginally better than the active control group on dMTS. In addition, although the n-back group improved more on the less difficult 2-back level than the more difficult 3-back level across training sessions, it was improvement on the 3-back level that accounted for 21% of the variance in dMTS performance. For the control group, improvement in training across sessions was unrelated to dMTS performance. At the neural level, greater activation in the left inferior frontal gyrus, right posterior parietal cortex, and the cerebellum distinguished the n-back group from the control group in the maintenance phase of dMTS. Degree of improvement on the 3-back level across training sessions was correlated with activation in right lateral prefrontal and motor cortices in the maintenance phase of dMTS. Our results suggest that although n-back training is more likely to improve performance in easier blocks, it is improvement in more difficult blocks that is predictive of performance on a target task drawing on WM. In addition, the extent to which training on a task can transfer to another task is likely due to the engagement of shared cognitive capacities and underlying neural substrates—in this case WM.N-back working memory (WM) tasks necessitate the maintenance and updating of dynamic rehearsal sets during performance. The delayed matching-to-sample (dMTS) task is another WM task, which in turn involves the encoding, maintenance, and retrieval of stimulus representations in sequential order. Because both n-back and dMTS engage WM function, we hypothesized that compared to a control task not taxing WM, training on the n-back task would be associated with better performance on dMTS by virtue of training a shared mental capacity. We tested this hypothesis by randomly assigning subjects ( Working memory (WM) can be defined as “a multicomponent system for active maintenance of information in the face of ongoing processing and/or distraction” , p. 770.An important factor that might affect transfer is the goodness-of-fit between the specific capacity enhanced during training and the cognitive requirements of the untrained activity. For example, Consistent with these process-specific findings, n denotes the specific number of trials that separate the current trial from the target trial. This task necessitates both maintenance and updating of dynamic rehearsal sets during performance . Specifically, during encoding participants memorize the stimulus, during maintenance they maintain the stimulus in WM, and during retrieval they press the button corresponding to the stimulus that matches the stimulus presented during encoding. Importantly, both n-back and dMTS are considered to be WM tasks (Our target WM task consisted of the delayed matching-to-sample (dMTS) task, a classic measure of short-term visual WM from the animal learning and WM literatures . dMTS inWM tasks , althougImportantly, n-back training could also impact the encoding and/or retrieval phases of dMTS because both tasks likely share those sub-processes beyond maintenance alone. For example, although there are explicit and compartmentalized encoding and maintenance phases within dMTS, the updating function inherent in the n-back very likely requires the encoding of memory representations as well as their retrieval for making matching decisions.In order to pinpoint the locus of transfer-related brain activity, we used fMRI to determine the phase within dMTS wherein activation would distinguish the experimental and control groups. Specifically, if as hypothesized training on the n-back task were to confer an advantage to dMTS performance by virtue of improving the maintenance function within WM, then one should observe a neural difference between the two groups in the maintenance phase. Furthermore, the differences between the two groups during the maintenance phase should be apparent in regions known to underlie delay-period maintenance in visual WM, including primarily the dorsolateral and ventrolateral prefrontal cortex , the inferior parietal lobule (BA 40) and adjacent parietal regions see . ConsistM = 30.76 years, SD = 9.71) with normal or corrected-to-normal vision. They were assigned randomly to the experimental (N = 22) or active control group (N = 21). To ensure similar expectations and motivations, participants were not informed about the existence of the two training conditions, or our hypotheses about the differential effects of training on outcome measures of interest (see 2 (1) = 0.01, p = 0.94], age , or fluid intelligence —assessed by administering the 18 even or odd items of Raven’s Advanced Progressive Matrices  were neurologically healthy right-handed  volunteerest see . There wMatrices  within aMatrices .Cognitive Test Software . We did not control for interference lures. Each block contained 150 trials. On 50 trials within each block the presented letter matched the target letter presented two or three positions earlier in the sequence (depending on the block), whereas on the remaining 100 trials it did not. Each letter was presented for 500 ms. Inter-stimulus interval (ISI) was a blank screen presented for 2500 ms. Participants pressed the spacebar when they detected a match.Participants in the active control group completed the 4-choice RT task . On eachFigure 1).Participants completed the dMTS in the fMRI scanner 3.29 days (SD = 1.11) after the last training session  = 2000 ms; echo time (TE) = 30 ms; flip angle (FA) = 70°; field of view (FOV) = 200 mm; 64 × 64 matrix; voxel dimensions = 3.1 mm × 3.1 mm × 5.0 mm], positioned to cover the whole brain. The first five volumes were discarded to allow for T1 equilibration effects. The number of volumes acquired was 354.A 3-Tesla MR scanner with an 8-channel head coil  was used to acquire T1 anatomical volume images (0.86 mm × 0.86 mm × 1.0 mm voxels). For functional imaging, T2∗ volumes, and spatially smoothed with an 8 mm full-width at half-maximum isotropic Gaussian kernel. Time series across each voxel were high-pass filtered with a cut-off of 128 s, using cosine functions to remove section-specific low frequency drifts in the BOLD signal. Condition effects at each voxel were estimated according to the GLM and regionally specific effects compared using linear contrasts. The BOLD signal was modeled as a box-car, convolved with a canonical hemodynamic response function.Data were analyzed using Statistical Parametric Mapping (SPM8). Head movement was less than 2 mm. All functional volumes were spatially realigned to the first volume. A mean image created from realigned volumes was spatially normalized to the MNI EPI brain template using non-linear basis functions. The derived spatial transformation was applied to the realigned T2http://afni.nimh.nih.gov/pub/dist/doc/manual/AlphaSim.pdf) incorporated in REST  within the avg152T2.nii whole-brain mask from the SPM toolbox. The real smoothness in the three directions was estimated from the residuals . Within our mask, the Monte Carlo simulations determined that a FWE-corrected false-positive probability of p < 0.05 was achieved using a voxel-wise threshold of p < 0.005 combined with a spatial extent threshold of 249 voxels.We applied a combination of voxel-height and cluster extent correction for multiple comparisons using AlphaSim , participants are considered to display good sensitivity, whereas when d′ is negative participants are incorrectly judging matches as mismatches and vice versa. In addition, we also investigated the effects of the two independent variables on the criterion—defined as the value of the decision variable deemed sufficiently high to determine that there is a match. A liberal value for the criterion biases the participant toward responding that there is a match, whereas a conservative value biases the participant toward responding that there is no match.For the experimental group we conducted a repeated-measures ANOVA with session  and level  as within-subjects variables. The key dependent variable was d′ improved across sessions, F = 10.50, p < 0.001, d′ was higher at sessions 2 and 3. There was no difference between sessions 2 and 3 (p = 0.10). There was also a main effect for level, demonstrating that d′ was greater on 2-back than 3-back, F = 25.05, p < 0.001, d′ improved more for 2-back than 3-back, F = 5.90, p < 0.01, Figure 2). In contrast, when we focused on the criterion as the dependent variable, the effects of session, level and the session × level interaction were not significant .For the experimental group, there was a main effect for session, demonstrating that M = 96.57%, SD = 3.10), 2 , and 3 , F = 1.48, p = 0.25, M = 0.45 s, SD = 0.06), second  and third sessions , F = 15.13, p = < 0.001, For the active control group we conducted an ANOVA with session  as the within-subjects variable, and accuracy as the dependent variable. As predicted see , performM = 96.70%, SD = 4.72) performed marginally better than the active control group  on dMTS, t(41) = 1.91, p = 0.06, Cohen’s d = 0.58. To directly test whether performance on dMTS would be a function of improvement in training on the n-back, for the experimental group we computed a new variable that was the difference in d′ between session 1 and session 3 (d′difference = d′session 3 -d′session 1)—separately for 2-back and 3-back. Next, we regressed accuracy (%) in dMTS performance onto d′difference. The results demonstrated that degree of improvement in 2-back was unrelated to dMTS performance, β = 0.31, p = 0.16. In contrast, degree of improvement in 3-back predicted variation in dMTS performance, β = 0.46, p = < 0.05. This result demonstrates that the degree of improvement in 3-back is a significant factor in dMTS performance. In fact, improvement in 3-back performance during training accounted for 21% of the variance in dMTS performance .The experimental group (difference = RTsession 3 - RTsession 1). Next, we regressed accuracy (%) in dMTS performance onto RTdifference. Importantly, only 19 data points (rather than 21) were included in this analysis because one participant failed to complete the third session of training, and another data point was excluded because it was an outlier—determined by its deviation from the means of both distributions by approximately 3 SDs .To determine whether the degree of improvement in 4-choice RT was predictive of dMTS performance amongst participants in the control group, we computed a new variable that was the difference in RT between session 1 and session 3 (RT SDs see . Degree Table 1 lists the regions activated in the encoding (-ITI), maintenance (-ITI), and retrieval (-ITI) phases of dMTS across all participants. An independent-samples t-test demonstrated greater activation in the n-back than active control group in the maintenance phase in the left inferior frontal gyrus , the right posterior parietal cortex , and the cerebellum . Neither the reverse contrast nor the contrasts in either direction involving the encoding or retrieval phase revealed any significant difference between the two groups. In other words, the difference in brain activation between the n-back and active control groups was limited exclusively to the maintenance phase of dMTS.Using an event-related design, we specified six regressors corresponding to (1) encoding, (2) maintenance, (3) retrieval, (4) ISI, (5) ITI, and (6) motor response. ISI and motor response were modeled out of the analyses by assigning weights of 0 to their corresponding regressors in all analyses. Figure 3A). To explore this effect at the neural level, we conducted three separate regression analyses to see whether difference in d′ for 3-back (d′difference = d′session 3 -d′session 1) would covary with brain activation during (1) encoding, (2) maintenance, and (3) retrieval. The results demonstrated that brain activation did not covary in relation to d′difference during encoding or retrieval. In contrast, during the maintenance phase brain activation in right lateral PFC  and motor cortex  covaried with d′difference .The analysis of our behavioral data had demonstrated that improvement in 3-back performance during training accounted for 21% of the variance in dMTS performance (d′ for 2-back (d′difference = d′session 3 -d′session 1) would covary with brain activation during (1) encoding, (2) maintenance, and (3) retrieval. Demonstrating a pattern similar to 3-back, brain activation did not covary in relation to d′difference during encoding or retrieval. However, during the maintenance phase d′difference covaried with activation in a distributed network in the brain, including three locations in right , left , and medial  middle frontal gyrus (BA 6), left lateral PFC , right superior parietal lobule , right cingulate , right extrastriate cortex , and three locations in left  and right  cerebellum .Although our behavioral data had demonstrated that for the experimental group improvement in 2-back performance was unrelated to dMTS performance, we nevertheless explored this effect at the neural level. As with 3-back, we conducted three separate regression analyses to see whether difference in difference = RTsession 3 - RTsession 1) would covary with brain activation during (1) encoding, (2) maintenance, and (3) retrieval. Our results demonstrated that there was no relationship between brain activation and RTdifference during encoding, maintenance or the retrieval phase.Finally, although our behavioral data had demonstrated that for the control group improvement in the 4-choice RT task was unrelated to dMTS performance, we nevertheless explored this effect at the neural level as well. Specifically, we conducted three separate regression analyses to see whether difference in RT in the 4-choice RT task , it was their degree of improvement in the 3-back level that predicted variation in dMTS performance, accounting for 21% of the observed variance in dMTS performance . Critically, degree of improvement in the 4-choice RT task in the control condition was unrelated to dMTS performance or its neural correlates, despite the fact that both are visuospatial tasks. These results demonstrate a dissociation between how training-related improvement in a WM task vs. a non-WM task is related to a target WM task. More specifically, they suggest that although performance on relatively easier levels of n-back is more likely to improve within three brief practice sessions, it is improvement in the more difficult levels that is more likely to be positively related to performance on target tasks drawing on the same capacity.The n-back group performed marginally better than the active control group on dMTS, registering a medium effect size . ImportaIn addition, the neural difference between the two groups was only apparent during the maintenance phase of dMTS, and localized to the left IFG, right PPC and the cerebellum. Sustained activation in the PFC has been related to maintenance in memory . Indeed,Figure 3A), we explored this effect at the neural level. Our results revealed that during the maintenance phase of dMTS, brain activation in right lateral PFC and motor cortex covaried with training-related improvement in 3-back . This region of the lateral PFC corresponds to Brodmann Area 44, and has been shown to be involved in both the storage and manipulation aspects of WM . Within this network, the frontal and parietal regions represent well-established nodes in the fronto-parietal WM network .In addition, although behaviourally training-related improvement in 2-back was unrelated to dMTS performance, our analyses of fMRI data demonstrated that during the maintenance phase of dMTS brain activation in a distributed network including the middle frontal gyrus, lateral PFC, superior parietal lobule, cingulate, extrastriate cortex, and the cerebellum covaried with training-related improvement in 2-back , gain data alone cannot be used as evidence for inferring transfer effects  following training. As such, our results are correlational, and we cannot draw causal inferences. In addition, although the degree of training-related improvement in 3-back predicted and accounted for 21% of the variance in dMTS performance . Doubling the matrix dimensions  reduces average accuracy rates to around 70% . In turn, this might have made it more difficult to observe differences between the two training conditions on this task, given that there was less room for improvement. There are at least two ways to increase the difficulty level on dMTS. First, on each trial we used a stimulus consisting of a 4 × 4 matrix (ound 70% . Second,Our results demonstrated that a group training on the n-back task performed marginally better than an active control group on dMTS. Although the n-back group improved more on 2-back than 3-back across three training sessions, it was improvement in 3-back that predicted and accounted for 21% of the variance in dMTS performance. There was no relationship between training-related gains and dMTS performance in the control group. At the neural level, the n-back group exhibited greater activation in the left IFG, right PPC and the cerebellum during the maintenance phase within dMTS. In addition, degree of improvement in 3-back covaried with brain activation in the right lateral prefrontal and motor cortices during the maintenance phase of dMTS, as did the degree of improvement in 2-back and activation in a distributed network including fronto-parietal WM nodes. In contrast, in the control group no relationship was observed between degree of improvement on the 4-choice RT task and dMTS performance. Combined, our results suggest that n-back training is more closely associated with dMTS performance than training on a task that does not tax WM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.